The present invention relates to the isolation and the characterization of two proteins having novel enzymatic activity, i.e. a porphyranase activity. These proteins are useful for hydrolyzing polysaccharides containing sulfated agaro-colloids and for producing oligo-porphyrans, notably oligo-porphyrans of defined structure and size.
Polysaccharides such as agars (agarose, porphyran, etc.) and carrageenans are very widely used as gelling agents or thickeners in various business sectors, notably in the agri-food and cosmetic industry. Thus, about 6,000 tons of agars and 22,000 tons of carrageenans are yearly extracted from red seaweed for agri-food purposes. The agars industrially produced from red algae of the genera Gelidium and Gracilaria. Moreover, the red alga Porphyra (known under the common name of nori) is the most consumed alga worldwide. The yearly production is estimated to be 90,000 tons/year (dry weight), representing a market of about 1.5 billion US dollars.
Polysaccharides and their derivatives are also of interest in the therapeutic field. Thus, enoxaprin (Lovenox®), which is marketed for treating thromboembolic disorders, is a mixture of chains of sulfated polysaccharides with variable lengths and formed with recurrent disaccharide units. Moreover, certain polysaccharide and oligosaccharide sulfates, such as oligo-fucan sulfates produced from algal polysaccharides, are useful as anti-microbial and/or anti-viral agents in humans (Ghosh et al. 2009 Glycobiology. 19:2-15).
Agaro-colloids are complex polysaccharides. Certain species of algae contain an agaro-colloid given as a majority compound, such as for example the alga Porphyra, which contains as main constituent the agaro-colloid called porphyran. Misnomeringly, polysaccharides extracted from red algae are sometimes also designated as porphyran, although this is in fact a mixture of agarose and of porphyran, porphyran being the majority compound.
The base disaccharide unit forming porphyran consists of a D-galactose unit (unit G, see FIG. 1) bound in β-1,4 to an L-galactose unit modified by O-sulfatation in position O6 (unit L6S, see FIG. 1). The disaccharide units are then connected together through α-1,3 bonds. This sulfated polysaccharide is commonly considered as the precursor of agarose. In a natural medium, other chemical modifications such as methylation, give rise to even more variations of this basic structure. These chemical modifications have the consequence of modulating the gelling nature of the polymer.
Up to now, enzymes capable of degrading agars are known and mainly used: α-agarases and β-agarases. β-agarases act on the β-1,4 bond (Jam et al. Biochem J. 2005 385:703-13) and α-agarases on the α-1,3 bond (Flament et al. Appl. Environ. Microbiol. 2007 73:4691-94) of the agarose disaccharide unit, and these enzymes are specific of the non-substituted or non-modified units. Indeed, there exist many bacteria, essentially marine bacteria which produce enzymes capable of hydrolyzing agars (Michel at al., Appl. Microbiol. Biotechnol. 2006 71:23-33). Several genes of β-agarases have already been cloned from these microorganisms, and the corresponding proteins over-expressed and purified.
More particularly, the first fully biochemically and structurally characterized β-agarases are produced by a bacterial strain isolated from the red alga Delesseria sanguinea (Jam et al. Biochem. J. 2005 385:703-13, Allouch et al. J. Biol. Chem. 2003 278:47171-80). This bacterial strain was deposited at the DSMZ (Deutsche Sammlung von Mikroorganismen and Zelikulturen GmbH) collection on May 8, 1998 under number DSM 12170. Taxonomic identification of this strain shows that it defines a new genus in the class of Flavobacteria and its characterization earned it the name of Zobellia galactanivorans (Barbeyron et al. Int. J. Syst. Evol. Microbiol. 2001 51:985-97). κ-carrageenase of this marine bacterium was also cloned and characterized (Barbeyron et al. Mol. Biol. Evol. 1998 15:528-37).
On the other hand, no specific enzyme of sulfated polysaccharides such as porphyran has yet been described to this day.
Misnomeringly, certain enzymes capable of degrading polysaccharides extracted from red algae containing porphyran were incorrectly designated as porphyranases in the scientific literature. However, these enzymes actually have agarase activity and degrade the agars present in the polysaccharides extracted from red algae.
Thus, Lee et al. (2006 Annual Meeting and International Symposium of the Korean Society for Microbiology and Biotechnology) describe the cloning and the sequencing of an enzyme of Vibrio pelagius, which is called porphyranase. However, this enzyme hydrolyzes D-galactoside β-1,4 bonds and leads to the obtaining of oligo-agroses, such as neoagarotetraose, neoagarohexaose and neoagarooctaose. It therefore has agarase activity and not porphyranase activity.
Similarly, Hatada et al. (2006 J. Agric. Food Chem. 54:9895-9900) mention β-agarases and indicate that the latter would have a <<porphyranase>> activity. However these enzymes are described of being capable of hydrolyzing D-galactoside β-1,4 bonds and of leading to the obtaining of agarose oligomers. These enzymes are therefore actually agarases and not porphyranases.
Also, Aoki et al. (2002 Marine and Highland Bioscience Center Report, 14:33-41) describe the cloning of a gene coding for a so-called porphyranase of Pseudomonas sp. ND 137. However, the fact that the corresponding protein degrades porphyran has never been demonstrated, and its strong sequence identity (40%) with the agarase of Pseudomonas sp. CY24 proves the contrary. In fact, the sequence of the protein cloned by Aoki et al. is annotated as an agarase and not as a porphyranase in entry number BAB79291.1 of the NCBI data base.
Certain degradation products of porphyran (oligo-porphyrans) were able to be obtained by chemical hydrolysis (Zhao et al. 2006 Int. J. Biol. Macromol. 38:45-50). This method however leads to the obtaining of a highly heterogeneous mixture of oligo-porphyrans. Further, hydrolysis is complete and the average molecular weight of the obtained mixture remains high.
Therefore there is a need for enzymes capable of catalyzing the hydrolysis of sulfated polysaccharides such as porphyran, and this in order to obtain novel agents which may be used in the agri-food, cosmetic and/or pharmaceutical industry.